Our research efforts are directed toward understanding the mechanism and consequences of Ty element retrotransposition. Retrotransposons are a class of transposable elements that resemble retroviruses, such as HIV-1, in their structure and mode of replication. Ty element relatives also comprise a significant fraction of mammalian and plant genomes. Ty elements are a paradigm for studying many aspects of retrotransposition because these elements are found in Saccharomyces cerevisiae, a highly developed eukaryotic model system. Eukaryotic genomes contain potentially unstable sequences whose rearrangement threatens genome structure and function. Here we show that certain mutant alleles of the nucleotide excision repair (NER)/TFIIH helicase genes RAD3 and SSL2 (RAD25) confer synthetic lethality, and destabilize the Saccharomyces cerevisiae genome by increasing both short sequence recombination and Ty1 retrotransposition. The rad3-G595R and ssl2-rtt mutations do not markedly alter Ty1 RNA or protein levels, or target site specificity. However, these mutations cause an increase in the physical stability of broken DNA molecules and unincorporated Ty1 cDNA, which leads to higher levels of short sequence recombination and Ty1 retrotransposition. We have also examined the role of the cellular homologous recombination functions on Ty1 retrotransposition. We find that transposition increases in cells mutated for genes in the RAD52 recombinational repair pathway, but not in cells mutated in DNA repair functions dedicated to mismatch repair (MSH2) or NER (RAD1 or RAD2). Like the NER/TFIIH mutants, the increase in Ty1 retrotransposition in mutants of the RAD52 group is correlated with a marked increase in the level of Ty1 cDNA. Together, our results link components of the core NER/TFIIH complex and functions required for homologous recombination/DNA double-strand break repair with genome stability, and host defense against Ty1 retrotransposition via a mechanism that involves DNA degradation. We have shown that, as in retroviruses, the genomic RNA in Ty1 virus-like particles (VLPs) is dimeric. The Ty1 dimers also resemble retroviral dimers in that they are stabilized during the proteolytic maturation of the VLP. The stabilization of the dimer suggests that one of the cleavage products of TyA1 possesses nucleic acid chaperone activity, and therefore, is functionally equivalent to the retroviral nucleocapsid (NC) protein. Interestingly, the thermostability of retroviral and Ty1 dimers is similar, yet examination of a 285-base cis-acting region of Ty1 RNA that is evidently sufficient for some transposition in the presence of a helper Ty1 element, has not revealed a kissing loop motif present in retroviral RNA. Furthermore, TyA1 lacks a recognizable NC domain, which has nucleic acid chaperone activity. These features suggest that packaging of Ty1 RNA occurs by a unique mechanism. The Ty1 retrovirus-like mobile genetic element transposes to new genomic locations via the element-encoded integrase (IN) protein. We report that purified recombinant IN is sufficient for correct integration of a linear DNA into a supercoiled target plasmid. Ty1 VLPs integrate donor DNA more efficiently than IN. VLP and IN-mediated insertions occur at random sites in the target. Magnesium is preferred over manganese for correct integration, and neither cation enhances nonspecific nuclease activity of IN. Products consistent with correct integration events have also been obtained by Southern analysis. Recombinant IN and VLPs utilize many, but not all, linear donor fragments containing non-Ty1 ends, including a U3 mutation which has been shown to be defective for transposition in vivo. Together, our results suggest that IN is sufficient for Ty1 integration in vitro and IN interacts with exogenous donors less stringently than with endogenous elements.